Functionalized graphene oxide nanosheets with folic acid and silk fibroin as a novel nanobiocomposite for biomedical applications

In this paper, a novel graphene oxide-folic acid/silk fibroin (GO-FA/SF) nanobiocomposite scaffold was designed and fabricated using affordable and non-toxic materials. The GO was synthesized using the hummer method, covalently functionalized with FA, and then easily conjugated with extracted SF via the freeze-drying process. For characterization of the scaffold, several techniques were employed: Fourier-transform infrared (FT-IR), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX), and thermogravimetric analysis (TGA). The cell viability method, hemolysis, and anti-biofilm assays were performed, exploring the biological capability of the nanobiocomposite. The cell viability percentages were 96.67, 96.35 and 97.23% for 24, 48, and 72 h, respectively, and its hemolytic effect was less than 10%. In addition, it was shown that this nanobiocomposite prevents the formation of Pseudomonas aeruginosa biofilm and has antibacterial activity.

. There is a broad spectrum of bioapplications of GO and its derivatives, including pH-dependent drug delivery, biosensing, bioimaging, and bone and skin tissue engineering [25][26][27][28][29][30][31][32] . GO can be combined with synthetic or natural materials such as polymers, proteins, and vitamins to improve its imperfections, e.g., tensile strength, elasticity, and conductivity. Folic acid (FA), a water-soluble vitamin, is commonly used in biomaterial with potential bioapplications such as imaging systems 33,34 , therapeutic agents 35 , and drug carriers 36 due to its low cost, compatibility in a biological medium, and non-toxicity 37 . Conjugation of FA to GO platform via amidation led to the preparation of biocompatible materials with a wide range of applications from biosensors to targeted delivery of therapeutic agents for cancer treatment, including breast, ovarian, lung, and colon cancers [38][39][40] .
Silk fibroin (SF) of silkworms is a well-known natural protein consisting of fibroin and sericin. The first one structurally constructs the silk fibers, and the second acts as a glue and binds the fibroin fibers together 41 . For centuries SF was used as a suture, and nowadays, it is considered a potential candidate for biomaterials and scaffolds for tissue engineering since it has significant mechanical strength, elasticity, biocompatibility and biodegradability [42][43][44] . In comparison with other proteins, silk has superiority such as low risk of infection, low-cost processing, easy isolation and purification, large scale availability, excellent mechanical properties, biodegradation and above all the presence of easily accessible chemical groups for functional modifications with preserving its advantageous intrinsic properties [45][46][47][48][49] .
Nanobiomaterials have been reported to show favorable potential in different bioapplications including nanomedicine 50 , pharmacology 51 , and biomedical fields. Despite the prominent advantages of nanobiomaterials, certain challenges exist in their applications. For instance, they may cause notable biological side effects such as an inflammatory reaction and eventual cell death 52 . To utilize the bioapplications of these nanomaterials, their toxicity and interactions with biological systems must be evaluated and considered. Owing to these issues, in this study GO was synthesized using hummer method, then it was functionalized with FA through covalent bonds and finally to improve its durability and prepare a composite it was blended with SF to construct final nanobiocomposite (GO-FA/SF) with predicted properties such as biocompatibility, and non-toxicity.

Experimental
Materials. Except for silkworm cocoons, the chemical reagents including dimethyl sulfuric acid, powdered graphite, potassium permanganate, anhydrous sodium carbonate, sodium nitrate, lithium bromide, N,N′dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), hydrogen peroxide, sodium chloride and the dialysis membrane (molecular weight cut-off = 14 kDa) were purchased from chemical Sigma-Aldrich company. All solvents used in this study and the fuming hydrochloric acid (37%) were also obtained from Merck company. The microorganism strain Pseudomonas aeruginosa (ATCCR 27853TM) was provided from Persian Type Culture Collection (PTCC, Karaj, Iran). Roswell Park Memorial Institute 1640 growth medium (RPMI) was purchased from Gibco BRL Life Technologies.
Instrumentation. Fourier-transform infrared (FT-IR) spectra were recorded on a Shimadzu FT-8400 s model, Japan spectrometer in the region 400-4000 cm −1 using KBr pellets. The morphology characteristic, size distribution, and percentage elemental analysis of samples were conducted via field emission-scanning electron microscopy (FE-SEM); (ZEISS-Sigma VP model, Germany) operating at a 15 kV. Before the examination, samples were mounted onto a metal stub using double-sided carbon adhesive tape and covered with a thin layer of gold, with the aid of a direct current sputter technique (Agar Sputter Coater model, Agar Scientific, England). Furthermore, the sample's elemental composition was characterized by an energy-dispersive X-ray (EDX) device (SAMx model, France). Besides, the distribution pattern of structural elements was determined by elemental mapping images. The X-ray diffraction (XRD) pattern was analyzed using the Brucker X-ray diffractometer device (D8 Advanced Model, Germany), which was equipped with Cu-Kα radiation (λ = 0.154 nm, 40 kV, 40 mA). The thermogravimetric analysis (TGA) was conducted using Bahr-STA 504 instrument (Germany) under the argon atmosphere. The adsorption of the samples was measured using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA) in the process of investigating the biological properties 53 .
Synthesis of GO-FA conjugate. According to our previous works, GO was prepared from native graphite flake and used the modified hummer method 23,24,32,54 . To activate the terminal carboxyl and carbonyl groups of GO, 1.85 g DCC and 0.3 g DMAP were added to 0.5 g of GO suspension in 100 mL DMSO. The mixture was sonicated for 20 min. In the next step, 0.3 g FA was added to the activated GO, and the suspension was sonicated for a further 20 min, and the obtained mixture was stirred overnight. After that, the mixture was centrifuged and washed two times with distilled water and two times with ethanol to remove DMSO and any nonconjugated FA from GO-FA. Finally, the mixture was washed with acetone and dried at room temperature to obtain powder product.
Preparation of GO-FA/SF nanobiocomposite. SF has been extracted from cocoons based on the previously reported methods through degumming, rehydration, and dialysis steps 55,56 . The FA decorated GO (GO-FA) (0.2 g) was sonicated in distilled water for a few minutes, and it was mixed with SF solution (5 mL). Later, the mixture solution was kept under stirring conditions for 4 h. Afterward, the obtained suspension mixture was poured into a petri dish for a further freeze-drying process. For this purpose, the sample was kept in a freezer (−70 °C) for 24 h. Afterward, the petri dish was placed at a freeze dryer device to sublimate the solvent for 24 h, and the dehydration process was performed at −60 °C with a constant pressure (0.1 bar) 57  www.nature.com/scientificreports/ MTT assay. First, 50 mg of Graphene Oxide-Folic Acid/Silk Fibroin (GO-FA/SF) nanobioocomposite was dispersed in 1 ml RPMI (Roswell Park Memorial Institute) medium using a shaker incubator for 24 h at 37 °C to obtain nanobiocomposite extract. Next, an MTT assay was performed to determine the toxicity of the GO-FA/ SF nanobiocomposite. For this purpose, the Hu02 cell line (human skin fibroblast cells) obtained from the Pasteur Institute of Iran cell bank was cultured in 96-well plates (1 × 10 5 cell/well) at optimal conditions (37 °C, 5% CO 2 in humidified incubator). Next, the growth media (10% FBS) was removed, and the cells were washed twice with Phosphate-buffered saline (PBS). New maintenance RPMI medium including GO-FA/SF extract was added and the cells were incubated for 24, 48, and 72 h. Attached RPMI without nanobiocomposite extract and cells in each well were also considered a negative control. A 10 μL solution of freshly prepared 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) in PBS was added to each well and allowed to incubate at 37 °C for 4 h. After that, the media with MTT solution was removed, and isopropanol was added at 100 µL/well. Next, the plates were shaken gently to facilitate formazan crystal solubilization 58 . The absorbance was measured at 590 nm using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA). Finally, the percentage of cell toxicity and cell viability was calculated as follows 59 : Hemolysis assay. This study was performed following the principles outlined in the declaration of Helsinki. Also, the experimental methods and the procedure for taking informed satisfaction were approved by Anti-biofilm assay. Tissue

Results and discussion
To prepare nano-sized GO-FA/SF biocomposite, the GO nanoflakes, FA vitamin, and fresh SF protein were employed. Four synthetic steps carried out the synthesis procedure. First, GO was synthesized using the modified hummer method. Second, it was functionalized with FA through coupling carboxylic acid of GO with the amine terminal of FA. Third, SF protein was extracted from silkworm cocoons via the degumming method. Finally, the surface-functionalized GO (GO-FA) blended with extracted SF using a freeze-drying process. The whole synthesis procedure is depicted in Fig. 1.

Characterization of the GO-FA/SF scaffold.
After the synthesis and preparation of the final nanobiocomposite, its structural characterization was also conducted using various techniques, including FT-IR, FE-SEM, EDX, XRD, and TGA. In the following section, each analysis is discussed as well.
(1) Toxicity% = 1 − mean OD of sample mean OD of control × 100 (2) Viability% = 100 − Toxicity%  Fig. 2A), a broad band at the region of 3400-3350 cm -1 was assigned to the stretching vibration mode of hydroxyl groups (-OH) 65 . Two distinct absorption bands at 1728 and 1622 cm -1 are attributed to the stretching vibration modes of the carboxyl group (C=O) and unoxidized graphitic domain (C=C), respectively 66 . Besides, small absorption bands related to the stretching vibration mode of alcohol groups (C-OH) and the stretching vibration mode of epoxy and alkoxy groups (C-O) were observed around 1222 and 1051 cm -167 . Concerning Fig. 2B, amide bond formation during FA conjugation could be characterized by the presence of the stretching vibration of (N-H) at the region of (3470-3320 cm -1 ), bending vibration of (N-H) at 1570 cm -1 and the stretching vibration of (C-N) of the aliphatic amine group at 1218 cm -168 . The characteristic peaks related to GO were observed at 1630, 1238 and 1045 cm -1 attributed to C=C, C-OH and C-OR vibrations respectively. Referring to the literature, it is evident that SF can be characterized by three vibrational bands in three regions including (1660-1625 cm -1 ), (1540-1520 cm -1 ), and (1270-1230 cm -1 ) related to (C=O) stretching vibration mode of amide I, the (N-H) bending vibration mode of amide II and the (C-N) stretching vibration mode of amide III, respectively 54,69 . With these in mind, the SF peaks mentioned above appeared at 1660 cm -1 (random coil), 1533 cm -1 (random coil), and 1272 cm -1 (β-sheet) confirmed the presence of SF threads in produced nanobiocomposite structure. As shown in Fig. 2C, assigning absorption bands at 1280 and 1078 cm −1 were attributed to the C-OH and C-OR stretching vibration modes of GO. Also, its better to mention that, the characteristic peaks of GO related to C=O and C=C vibrations were overlapped by sharp peaks attributed to SF random coil vibrations at the region of 1660-1533 cm −1 .
EDX analysis. The structural characterization of synthesized GO-FA and GO-FA/SF composites was carried out using energy dispersive x-ray spectroscopy to obtain the elemental composition. As depicted in Fig. 3A and www.nature.com/scientificreports/ B, the presence of two strong peaks related to carbon and oxygen elements and observing nitrogen signals confirm the preparation of GO and its functionalization with FA, respectively. Most importantly, the increase of the percentage of nitrogen content from 3.3 to 5.8 wt% (Fig. 3B) revealed that the SF was successfully conjuncted to GO-FA composite in the final step since SF brought extra nitrogen element with itself to final bionanocomposite.

FE-SEM imaging study.
The morphological characterization of starting GO and final GO-FA/SF nanobiocomposite was performed using field emission scanning electron microscopy FE-SEM. As shown in Fig. 4A, the layered structure of GO flakes with wavy morphology is obvious. Considering Fig. 4B, following the functionalization of GO sheets with FA chains and its modification with SF polymers, it is revealed that the modified GO displayed smooth unilamellar morphology with reduced ruggedness, while it was preserved its characteristic structure observed in the native GO flakes.
XRD pattern. X-ray diffraction (XRD) was used to investigate the crystal phase and determine the interlayer spacing for GO and final GO-FA/SF composite. As depicted in Fig. 5A, GO showed a sharp characteristic peak at 2θ = 10.7º (002) with the interlayer d spacing of 0.82 nm corresponding to GO sheets 70 . Figure 5B shows the semi-crystalline nature of GO-FA/SF composite as GO was functionalized with FA and loaded with SF. As illustrated in Fig. 5B, the well-defined peak of GO was observed at 2θ = 7.8º with increased interlayer d spacing of    www.nature.com/scientificreports/ Thermal study. The thermogravimetric analysis was carried out to inspect the thermal stability of the prepared GO-FA/SF nanobiocomposite. Figure 6 shows the mass loss of the sample as a function of temperature, and it is revealed that product composite degrades mainly in four steps. According to the published results, the first significant mass loss at almost 170-260 Cº (≈40%) is mainly due to the decomposition of oxygen-containing functional groups of GO such as epoxide, hydroxyl, and carboxyl groups, yielding CO and CO 2 [75][76][77] . The second mass reduction at a temperature range of 260-310 Cº (≈14%) corresponds to the decomposition of FA moieties that functionalized the GO platform 73 . So, we can calculate that the composite contains about 0.28 mg FA. The third degradation range at 310-455 Cº (≈12%) could be explained by cleavage of peptide bonds and breaking down of the side chain group of amino acids related to SF protein in nanobiocomposite structure 78,79 . Following the temperature increase to 800Cº, the mass loss by ≈34% refers to the decomposition of all of the remaining moieties with almost any residual.
Bio-application of GO-FA/SF bio-nanobiocomposite. Biocompatibility. As shown in Fig. 7, the viability percentages of Hu02 cells treated with GO-FA/SF nanobiocomposite, after 24 and 48 h of incubation were 96.67 and 96.35%, respectively. This value increased to 97.23% after 72 h of incubation. Also, the effect of GO-FA/SF nanobiocomposite on morphology and shape of Hu02 cells after 72 h of incubation was imaged with a reverse microscope. It's indicated that Hu02 cells retain their fibroblast shape after treatment with GO-FA/SF. Results are the average of three independent experiments. These results illustrate that GO-FA/SF nanobiocomposite had no toxic effects on Hu02 cells.
Hemocompatibility. The test results, which averaged the results of the three independent experiments, showed that the hemolytic effects of the nanobiocomposite extract after different extraction times (24 and 72 h) were below 10%, while 1% triton X-100 lysed almost all RBCs. (P ≤ 0.001) (Fig. 8). Based on the results, it can be said that GO-FA/SF nanobiocomposite is hemocompatible.
Anti-biofilm activity. As shown in Fig. 9, the adsorption rate of polystyrene (as a positive control) at 570 nm was 0.76, which was reduced to 0.63 for GO-FA/SF nanobiocomposite. So, it showed a small amount of antibiofilm effects. The reported values are the average of three independent repetitions of the experiment.

Conclusions
This study designed and fabricated a novel nanobiocomposite using non-toxic and inexpensive starting materials. GO was synthesized through the modified hummer method, functionalized with FA, and then easily conjugated with extracted SF to aim this goal. The series of biological tests revealed remarkable features for obtained GO-FA/ SF bionanocomposite. The MTT assay showed that treatment of Hu02 cells with GO-FA/SF nanobiocomposite for 24, 48, and 72 h of incubation time resulted in 96.67%, 96.35%, and 97.23% viability percentages with maintaining their fibroblast shape, which overall confirms the biocompatibility and non-toxicity of GO-FA/SF nanobiocomposite toward Hu02 cells. To explain the hemocompatibility of the produced nanobiocomposite, the hemolysis assay was performed, and the hemolysis percentage of 7.88 and 8.62% for 24 h and 72 h of extraction times were obtained. The results of the anti-biofilm assay showed that the fabricated nanobiocomposite scaffold could well inhibit the P. aeruginosa biofilm formation on its surface. Considering the structural characteristics